ZENK protein regulation by song in the brain of songbirds

When songbirds hear the song of another individual of the same species or when they sing, the mRNA levels of the ZENK gene increase rapidly in forebrain areas involved in vocal communication. This gene induction is thought to be related to long-term neuronal change and possibly the formation of song-related memories. We used immunocytochemistry to study the levels and distribution of ZENK protein in the brain of zebra finches and canaries after presentation of song playbacks. Birds that heard the playbacks and did not sing in response showed increased ZENK protein levels in auditory brain areas, including the caudomedial neostriatum and hyperstriatum ventrale, fields L1 and L3, the shelf adjacent to the high vocal center (HVC), the cup adjacent to the nucleus robustus archistriatalis (RA), and the nucleus mesencephalicus lateralis pars dorsalis (MLd). No ZENK expression was seen in song nuclei in these birds. Males that sang in response to the playbacks showed, in addition to auditory areas, increased ZENK protein in several song control nuclei, most prominently in HVC, RA, area X, and the dorsomedial nucleus (DN) of the intercollicular complex. The rise in ZENK protein followed that described previously for ZENK mRNA by a short lag, and the distribution of ZENK-labeled cells was in agreement with previous analysis of mRNA distribution. Thus, ZENK protein regulation can be used to assess activation of brain areas involved in perceptual and motor aspects of song. Possible implications of ZENK induction in these areas are discussed. J. Comp. Neurol. 393:426–438, 1998. © 1998 Wiley-Liss, Inc.     

Seasonal neuronal plasticity in song‐control and auditory forebrain areas in subtropical nonmigratory and palearctic‐indian migratory male songbirds

This study examines whether differences in annual life‐history states (LHSs) among the inhabitants of two latitudes would have an impact on the neuronal plasticity of the song‐control system in songbirds. At the times of equinoxes and solstices during the year (n = 4 per year) corresponding to different LHSs, we measured the volumetric changes and expression of doublecortin (DCX; an endogenous marker of the neuronal recruitment) in the song‐control nuclei and higher order auditory forebrain regions of the subtropical resident Indian weaverbirds (Ploceus philippinus) and Palearctic‐Indian migratory redheaded buntings (Emberiza bruniceps). Area X in basal ganglia, lateral magnocellular nucleus of the anterior nidopallium (LMAN), HVC (proper name), and robust nucleus of the arcopallium (RA) were enlarged during the breeding LHS. Both round and fusiform DCX‐immunoreactive (DCX‐ir) cells were found in area X and HVC but not in LMAN or RA, with a significant seasonal difference. Also, as shown by increase in volume and by dense, round DCX‐ir cells, the neuronal incorporation was increased in HVC alone during the breeding LHS. This suggests differences in the response of song‐control nuclei to photoperiod‐induced changes in LHSs. Furthermore, DCX immunoreactivity indicated participation of the cortical caudomedial nidopallium and caudomedial mesopallium in the song‐control system, albeit with differences between the weaverbirds and the buntings. Overall, these results show seasonal neuronal plasticity in the song‐control system closely associated with annual reproductive LHS in both of the songbirds. Differences between species probably account for the differences in the photoperiod‐response system between the relative refractory weaverbirds and absolute refractory redheaded buntings. J. Comp. Neurol. 524:2914–2929, 2016. © 2016 Wiley Periodicals, Inc.     

Expression of glutamate and inhibitory amino acid vesicular transporters in the rodent auditory brainstem

Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus, suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 coexpressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits.     

Distribution of vesicular glutamate transporters in the brain of the turtle (Pseudemys scripta elegans)

The distribution of glutamatergic neurons has been extensively studied in mammalian and avian brains, but its distribution in a reptilian brain remains unknown. In the present study, the distribution of subpopulations of glutamatergic neurons in the turtle brain was examined by in situ hybridization using probes for vesicular glutamate transporter (VGLUT) 1–3. Strong VGLUT1 expression was observed in the telencephalic pallium; the mitral cells of the olfactory bulb, the medial, dorsomedial, dorsal, and lateral parts of the cerebral cortex, pallial thickening, and dorsal ventricular ridge; and also, in granule cells of the cerebellar cortex. Moderate to weak expression was found in the lateral and medial amygdaloid nuclei, the periventricular cellular layer of the optic tectum, and in some brainstem nuclei. VGLUT2 was weakly expressed in the telencephalon but was intensely expressed in the dorsal thalamic nuclei, magnocellular part of the isthmic nucleus, brainstem nuclei, and the rostral cervical segment of the spinal cord. The cerebellar cortex was devoid of VGLUT2 expression. The central amygdaloid nucleus did not express VGLUT1 or VGLUT2. VGLUT3 was localized in the parvocellular part of the isthmic nucleus, superior and inferior raphe nuclei, and cochlear nucleus. Our results indicate that the distribution of VGLUTs in the turtle brain is similar to that in the mammalian brain rather than that in the avian brain.     

Functional evidence for internal feedback in the songbird brain nucleus HVC

The song control system of songbirds consists mainly of the 'motor pathway' and 'anterior forebrain pathway'. The medial magnocellular nucleus of anterior nidopallium (mMAN) projects to the song control nucleus HVC, which is the point of divergence of the two pathways. We made simultaneous multiunit electrophysiological recordings from the mMAN and HVC in anesthetized Bengalese finches. We confirmed that the mMAN neurons showed song-selective auditory responses, and found temporal correlations between song-related activities of the mMAN and HVC neurons. The temporal relationship between the neural activation of the HVC and mMAN suggests that these nuclei are parts of a closed loop, which could provide internal feedback to the HVC for sequential syllable control.     

Distributions of vesicular glutamate transporters 1 and 2 in the visual system of tree shrews (Tupaia belangeri)

Vesicular glutamate transporter (VGLUT) proteins regulate the storage and release of glutamate from synapses of excitatory neurons. Two isoforms, VGLUT1 and VGLUT2, are found in most glutamatergic projections across the mammalian visual system, and appear to differentially identify subsets of excitatory projections between visual structures. To expand current knowledge on the distribution of VGLUT isoforms in highly visual mammals, we examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the lateral geniculate nucleus (LGN), superior colliculus, pulvinar complex, and primary visual cortex (V1) in tree shrews (Tupaia belangeri), which are closely related to primates but classified as a separate order (Scandentia). We found that VGLUT1 was distributed in intrinsic and corticothalamic connections, whereas VGLUT2 was predominantly distributed in subcortical and thalamocortical connections. VGLUT1 and VGLUT2 were coexpressed in the LGN and in the pulvinar complex, as well as in restricted layers of V1, suggesting a greater heterogeneity in the range of efferent glutamatergic projections from these structures. These findings provide further evidence that VGLUT1 and VGLUT2 identify distinct populations of excitatory neurons in visual brain structures across mammals. Observed variations in individual projections may highlight the evolution of these connections through the mammalian lineage. J. Comp. Neurol. 523:1792–1808, 2015. © 2015 Wiley Periodicals, Inc.     

Reafferent thalamo-“cortical” loops in the song system of oscine songbirds

Songbirds have a complex vocal repertoire, much of which is learned by imitation. The vocal motor system of songbirds includes a set of telencephalic pathways dedicated to the acquisition and production of learned song. The main vocal motor pathway goes from the high vocal center (HVC) to the robust nucleus of the archistriatum (RA), which in turn innervates mesencephalic and medullary nuclei involved in vocalization. We used neural tract tracers (biotinylated dextran amines, fluorescein- and rhodamine-linked dextran amines, and Fluorogold) to show that RA of adult male canaries (Serinus canaria) and zebra finches (Taeniopygia guttata) sends an ipsilateral projection to the posterior portion of the dorsomedial thalamic nucleus (DMP). DMP projects to the medial portion of the magnocellular nucleus of the anterior neostriatum (mMAN), which is known to project to HVC, forming a feedback circuit. We also observed that the projection from DMP to mMAN is bilateral. Extracellular multi-unit recordings from awake restrained subjects have demonstrated that mMAN has auditory responses that are selective for the bird's own song. These auditory responses are similar to responses recorded simultaneously in HVC, but with a longer latency, suggesting that mMAN receives auditory information from HVC through the circuit we have described. We also saw a weaker projection from RA to the medial part of the dorsolateral nucleus of the thalamus (DLM), which is known to project to the lateral portion of the magnocellular nucleus of the anterior neostriatum (lMAN). lMAN is known to project to RA, completing yet another feedback circuit; lMAN is also part of the anterior forebrain pathway, which plays an essential role in song learning. These thalamo-telencephalic circuits are similar to thalamo-cortical circuits found in mammalian motor systems, and we suggest that the signals carried by these loops may be important for song perception, song learning, song production, and/or the bilateral coordination of vocal motor commands. J. Comp. Neurol. 380:275–290, 1997. © 1997 Wiley-Liss, Inc.     

Collapsin response mediator protein-4 (CRMP-4) expression in posthaching development of song control nuclei in Bengalese finches

CRMP-4 is regarded to play a role in neuronal differentiation, neurite growth and synapse formation. It has been shown to express in brain areas undergoing plastic changes or neuronal generation. Bird song is a learned, complex behavior. During song learning, some neural changes occur dramatically within song nuclei in neuron number, neuronal morphology, and synaptic formation or rearrangements. In order to get insights into the potential functions of CRMP-4 in the posthatching development of song nuclei during song learning, we examined the expression of CRMP-4 protein and mRNA in song control nuclei of Bengalese finch (Lonchura striata) from posthatching days (P) 10 to adulthood. Our study showed that cells positive for CRMP-4 protein and mRNA were distributed in song nuclei nearly in all the studied groups. The numbers of CRMP-4 cells in most of studied song nuclei changed significantly with age. They reached the peak at P15 in the lateral magnocellular nucleus of anterior nidopallium (LMAN) and the caudal medial nidopallium (NCM), or at P25 in HVC, Area X and the dorsolateral nucleus of the medial anterior thalamus (DLM). They then continued to decrease till adulthood. CRMP-4 protein and mRNA were both relatively high expressed during the post-hatch development of song control nuclei and song learning (P20-60), suggesting that CRMP-4 is involved in these activities. Although CRMP-4 protein and mRNA largely decreased at adulthood, they continued to express moderately, revealing that CRMP-4 may play a role in the maintenance of adult song nuclei.     

Anatomical plasticity in the adult zebra finch song system

In many songbirds, vocal learning‐related cellular plasticity was thought to end following a developmental critical period. However, mounting evidence in one such species, the zebra finch, suggests that forms of plasticity common during song learning continue well into adulthood, including a reliance on auditory feedback for song maintenance. This reliance wanes with increasing age, in tandem with age‐related increases in fine motor control. We investigated age‐related morphological changes in the adult zebra finch song system by focusing on two cortical projection neuron types that 1) share a common efferent target, 2) are known to exhibit morphological and functional change during song learning, and 3) exert opposing influences on song acoustic structure. Neurons in HVC and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) both project to the robust nucleus of the arcopallium (RA). During juvenile song learning and adult song maintenance, HVC promotes song syllable stereotypy, whereas LMAN promotes learning and acoustic variability. After retrograde labeling of these two cell types in adults, there were age‐related increases in dendritic arbor in HVC‐RA but not LMAN‐RA neurons, resulting in an increase in the ratio of HVC‐RA:LMAN‐RA dendritic arbor. Differential growth of HVC relative to LMAN dendrites may relate to increases in song motor refinement, decreases in the reliance of song on auditory feedback, or both. Despite this differential growth with age, both cell types retain the capacity for experience‐dependent growth, as we show here. These results may provide insights into mechanisms that promote and constrain adult vocal plasticity. J. Comp. Neurol. 520:3673–3686, 2012. © 2012 Wiley Periodicals, Inc.     

VGLUT‐VGAT expression delineates functionally specialised populations of vasopressin‐containing neurones including a glutamatergic perforant path‐projecting cell group to the hippocampus in rat and mouse brain

The origin and functional significance of vasopressin (AVP)‐containing fibres in limbic regions has been an ongoing subject of investigation for several years. We have previously identified AVP‐magnocellular neurones of rat hypothalamus that provide glutamatergic projections to the hippocampus, amygdala, lateral habenula and locus coeruleus. However, we also reported AVP‐immunopositive fibres in those regions that are thin and make Gray type II synapses, which are unlikely to be of magnocellular origin. Therefore, in the present study, we characterised AVP mRNA co‐expression with expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) neuronal traits in rat and mouse brain, using high‐resolution in situ hybridisation methods, including a radio‐ribonucleotide and RNAscope 2.5 HD duplex assay, with Slc17a7, Slc17a6, Slc32a1 and Avp probes corresponding to mRNAs of VGLUT1, VGLUT2, VGAT and AVP, respectively. We located 18 cell groups expressing Avp and identified their molecular signatures for VGLUT and VGAT mRNA expression. Avp cell groups of hypothalamus and midbrain are mainly VGLUT mRNA‐expressing, whereas those in regions derived from cerebral nuclei are mainly VGAT mRNA‐expressing, suggesting a functional segregation of glutamate/GABA co‐transmission with AVP. A newly identified Slc17a7 and Slc17a6 (but not Slc32a1) expressing vasopressinergic cell group was found in layer II‐III neurones of the central entorhinal cortex, which projects to the hippocampus. These data support the notion of a complex role for AVP with respect to modulating multiple central circuits controlling behaviour in specific ways depending on co‐transmission with glutamate or GABA, potentially giving rise to a functional classification of AVPergic neurones in the central nervous system.     

Vessicular glutamate transporters 1 and 2 are differentially associated with auditory nerve and spinal trigeminal inputs to the cochlear nucleus

Projections of glutamatergic somatosensory and auditory fibers to the cochlear nucleus (CN) are mostly nonoverlapping: projections from the spinal trigeminal nucleus (Sp5) terminate primarily in the granule cell domains (GCD) of CN, whereas type I auditory nerve fibers (ANFs) project to the magnocellular areas of the VCN (VCNm) and deep layers of Dorsal CN (DCN). Vesicular glutamate transporters (VGLUTs), which selectively package glutamate into synaptic vesicles, have different isoforms associated with distinct subtypes of excitatory glutamatergic neurons. Here we examined the distributions of VGLUT1 and VGLU2 expression in the CN and their colocalization with Sp5 and ANF terminals following injections of anterograde tracers into Sp5 and the cochlea in the guinea pig. The CN regions that showed the most intense expression of VGLUT1 and VGLUT2 were largely nonoverlapping and were consistent with ANF and Sp5 projections, respectively: VGLUT1 was highly expressed in VCNm and the molecular layer of the DCN, whereas VGLUT2 was expressed predominantly in the GCD. Half (47% +/- 3%) of the Sp5 mossy fiber endings colabeled with VGLUT2, but few (2.5% +/- 1%) colabeled with VGLUT1. In contrast, ANFs colabeled predominantly with VGLUT1. The pathway-specific expression of VGLUT isoforms in the CN may be associated with the intrinsic synaptic properties that are unique to each sensory pathway.     

Brain expression and song regulation of the cholecystokinin gene in the zebra finch (Taeniopygia guttata)

The gene encoding cholecystokinin (Cck) is abundantly expressed in the mammalian brain and has been associated with such functions as feeding termination and satiety, locomotion and self-stimulation, the modulation of anxiety-like behaviors, and learning and memory. Here we describe the brain expression and song regulation of Cck in the brain of the adult male zebra finch (Taeniopygia guttata), a songbird species. Using in situ hybridization we demonstrate that Cck is highly expressed in several discrete brain regions, most prominently the caudalmost portion of the hippocampal formation, the caudodorsal nidopallial shelf and the caudomedial nidopallium (NCM), the core or shell regions of dorsal thalamic nuclei, dopaminergic cell groups in the mesencephalon and pons, the principal nucleus of the trigeminal nerve, and the dorsal raphe. Cck was largely absent in song control system, a group of nuclei required for vocal learning and song production in songbirds, although sparse labeling was detected throughout the striatum, including song nucleus area X. We also show that levels of Cck mRNA and the number of labeled cells increase in the NCM of males and females following auditory stimulation with conspecific song. Double labeling further reveals that the majority of Cck cells, excluding those in the reticular nucleus of the thalamus, are non-GABAergic. Together, these data provide the first comprehensive characterization of Cck expression in a songbird, and suggest a possible involvement of Cck regulation in important aspects of birdsong biology, such as perceptual processing, auditory memorization, and/or vocal-motor control of song production.     

Glutamatergic neuronal populations in the brainstem of the sea lamprey,Petromyzon marinus: An in situ hybridization and immunocytochemical study

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium‐sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract‐tracing methods or γ‐aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium‐sized cells of some reticulospinal‐projecting groups were often glutamate‐immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal‐projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non‐glutamatergic cells or neurons that co‐release both GABA and glutamate. J. Comp. Neurol. 521:522–557, 2013. © 2012 Wiley Periodicals, Inc.     

Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat

VGLUT1 and VGLUT2 have been reported to show complementary distributions in most brain regions and have been assumed to define distinct functional elements. In the present study, we first investigated the expression of VGLUT1 and VGLUT2 in the trigeminal sensory nuclear complex of the rat by dual‐fluorescence in situ hybridization. Although VGLUT1 and/or VGLUT2 mRNA signals were detected in all the nuclei, colocalization was found only in the principal sensory trigeminal nucleus (Vp). About 64% of glutamatergic Vp neurons coexpressed VGLUT1 and VGLUT2, and the others expressed either VGLUT1 or VGLUT2, indicating that Vp neurons might be divided into three groups. We then injected retrograde tracer into the thalamic regions, including the posteromedial ventral nucleus (VPM) and posterior nuclei (Po), and observed that the majority of both VGLUT1‐ and VGLUT2‐expressing Vp neurons were retrogradely labeled with the tracer. We further performed anterograde labeling of Vp neurons and observed immunoreactivies for anterograde tracer, VGLUT1, and VGLUT2 in the VPM and Po. Most anterogradely labeled axon terminals showed immunoreactivities for both VGLUT1 and VGLUT2 in the VPM and made asymmetric synapses with dendritic profiles of VPM neurons. On the other hand, in the Po, only a few axon terminals were labeled with anterograde tracer, and they were positive only for VGLUT2. The results indicated that Vp neurons expressing VGLUT1 and VGLUT2 project to the VPM, but not to the Po, although the functional differences of three distinct populations of Vp neurons, VGLUT1‐, VGLUT2‐, and VGLUT1/VGLUT2‐expressing ones, remain unsettled. J. Comp. Neurol. 518:3149–3168, 2010. © 2010 Wiley‐Liss, Inc.     

Vocal control pathways through the anterior forebrain of a parrot (Melopsittacus undulatus)

A feature of the telencephalic vocal control system in the budgerigar (Melopsittacus undulatus) that has been hypothesized to represent a profound difference in organization from the oscine vocal system is its reported lack of an inherent circuit through the anterior forebrain. The present study reports anatomical connections that indicate the existence of an anterior forebrain circuit comparable in important ways to the “recursive” pathway of oscine songbirds. Results from anterograde and retrograde tracing experiments with biocytin and fluorescently labeled dextran amines indicate that the central nucleus of the anterior archistriatum (AAc) is the source of ascending projections upon the oval nuclei of the anterior neostriatum and ventral hyperstriatum (NAo and HVo, respectively). Efferent projections from the latter nuclei terminate in the lateral neostriatum afferent to AAc, thereby forming a short recurrent pathway through the pallium. Previously reported projections from HVo and NAo upon the magnocellular nucleus of the lobus parolfactorius (LPOm), and from LPOm onto the magnocellular nucleus of the dorsal thalamus (DMm; G.F. Striedter [1994] J. Comp. Neurol. 343:35–56), are confirmed. A specific projection from DMm onto NAom is also demonstrated; therefore, a recurrent pathway through the basal forebrain also exists in the budgerigar vocal system that is similar to the anterior forebrain circuit of oscine songbirds. Parallels between these circuits and mammalian basal ganglia-thalamo-cortical circuits are discussed. It is hypothesized that vocal control nuclei of the avian anterior neostriatum may perform a function similar to the primate supplemental motor area. J. Comp. Neurol. 377:179–206, 1997. © 1997 Wiley-Liss, Inc.     

Distribution of androgen receptor mRNA expression in vocal,auditory, and neuroendocrine circuits in a teleost fish

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive‐related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context‐dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain‐spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase‐containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. J. Comp. Neurol. 518:493–512, 2010. © 2009 Wiley‐Liss, Inc.     

Distribution of vesicular glutamate transporter mRNA in rat hypothalamus

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual-label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65-positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function.